Studies with protein kinase C delta knockout (PKC4-/-) mice indicate that PKC4 regulates ethanol intoxication and self-administration. These effects may be related to PKC4 actions at extrasynaptic GABAA receptors, since thalamic and hippocampal neurons from PKC4-/- mice lack ethanol enhancement of tonic inhibitory GABA currents. The main hypothesis of this project is that PKC4 regulates ethanol intoxication and self-administration by phosphorylating proteins that alter the function of extrasynaptic GABAA receptors in brain regions controlling these behaviors. This hypothesis will be tested using a novel mouse model, a knock-in mouse that expresses an ATP analog-sensitive mutant of PKC4 (AS-PKC4). AS-PKC4 can be selectively and potently inhibited by analogs of the general kinase inhibitor PP1 that cross the blood brain barrier and can be administered orally or by i.p. injection. Studies will determine if inhibition of AS-PKC4 mice increases signs of ethanol intoxication and enhances ethanol self-administration in adult mice. Electrophysiological studies in brain slices will investigate whether inhibiting AS-PKC4 blocks ethanol potentiation of tonic GABA currents in hippocampal, thalamic, and amygdala neurons. In vitro kinase assays will determine if 22, 23, and 4 subunits are possible substrates of PKC4. Novel ATP analogs will be used with tissues from AS-PKC4 mice to identify PKC4 phosphorylation sites on GABAA receptor-associated proteins. Studies in cells that heterologously express 1422/34 receptors and receptor-associated proteins with alanine substitutions at PKC4 phosphorylation sites, will determine if these sites regulate the ethanol sensitivity of 1422/34 receptors. The overall goal of this project is to identify a novel PKC4 signaling pathway that regulates the ethanol sensitivity of extrasynaptic GABAA receptors and may contain targets for the development of new therapeutics to treat alcohol use disorders. PUBLIC HEALTH RELEVANCE We will investigate the role of PKC4 in ethanol's effects on behavior and on regulation of GABAA receptors. These studies will utilize an ATP-analog sensitive form of PKC4 that can be inhibited with high selectivity and specificity, and can be used to identify PKC4 substrates. The results of these studies will reveal new molecular mechanisms that regulate GABA signaling, behavioral sensitivity to ethanol, and ethanol consumption.